ISTRIBUIÇÃO ENTRE AS BESORREGIÕES
ARTIGO CIENTÍFICO PARA PUBLICAÇÃO SEGUNDO AS NORMAS DA REVISTA FORENSIC SCIENCE INTERNATIONAL
15 STR loci frequencies in the population from Rio Grande do Norte, Brazil
Taissa Maria Moura de Oliveiraab, Andrea Luciana Cunha Fernandesa, Gioconda Dias Rodrigues Leãoa, Roberto Chaves de Vasconcelosa, Erica Aires Gila, Juliana Mendonça Freirea, Geraldo Barroso Cavalcanti Júniorb.
a Laboratório DNA Center. Av. Afonso Pena, 952, TIrol. Natal-RN. CEP: 59020-100. Brasil.
b Programa de Pós-graduação em Ciências Farmacêuticas – UFRN.
Email address: taissa_oliveira@oi.com.br
ABSTRACT
Allele frequencies for 15 Short Tandem Repeats loci (D8S1179, D21S11, D7S820, CSF1PO, D19S433, vWA, TPOX, D18S51, D3S1358, TH01, D13S317, D16S539, D2S1338, D5S818 e FGA) were obtained from a sample of 1100 unrelated individuals undergoing paternity testing. The population is from Rio Grande do Norte, Northeastern Brazil. The loci are the most commonly used in forensic and paternity testing, being analyzed by the AmpFlSTR® Identifiler (Applied Biosystems) commercial kit. The most polimorphic loci were D2S1338 and D18S51. Excepting the D21S11, D19S433 and D2S1338, all loci were in Hardy-Weinberg equilibrium. Comparative analyses between our population data and other populations are presented.
Introduction
Many forensic laboratories worldwide are evaluating and implementing highly polymorphic DNA loci, whose polymorphism derive from Short Tandem Repeated (STR) core sequences. Simultaneous amplification and subsequent typing of a number of these STR loci have significantly enhanced the capabilities of forensic scientists to analyze DNA derived from biological specimens [1]. It is well known that the use of Short Tandem Repeats for forensic purposes requires the existence of databases that reflect the allele distribution in the population they will be applied, that provides subsidies to improve the reliability of the results of determination of the genetic link [2]. Thus, when assessing the frequency of the microregion, a possible distortion of data is sent off. Our aim is to study the allele frequencies for 15 STR loci and calculate the statistical parameters of forensic interest, in a sample provenient from Rio Grande do Norte State, Brazil.
Population
The allelic frequency of 15 short tandem repeats - STR (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, D5S818, FGA) was evaluated in population from Rio Grande do Norte State, localized in the northeast region of Brazil. The current population is about 3 million inhabitants (according to IBGE, 2010), divided into four mesoregions, named: Oeste Potiguar, Cental Potiguar, Agreste Potiguar and Leste Potiguar.
Blood samples from 1,100 unrelated individuals were obtained from paternity testing cases conducted in each mesoregion of the State and shipped immediately to DNA Center Laboratory (Natal City) for analysis. This sample is representative of the population of Rio Grande do Norte State, and 25% of the individuals included are from the Oeste Potiguar mesoregion, 17% from the Central Potiguar, 16% from the Agreste Potiguar and 42% from the Leste Potiguar.
Donors were subjected to an interview in order to obtain their informed consent. DNA Extraction
DNA was extracted from blood spots on Whatman FTA cards using the manufacturer’s protocols.
PCR
A total of 0.5–1.0 ng of DNA, contained in a 1.2 mm circle of FTA paper, was used to simultaneous amplification of 15 STR loci (multiplex PCR) plus the gender determination marker, Amelogenin, performed using the AmpFlSTR® IdentifilerTM
PCR Amplification Kit (Applied Biosystems, Foster City, CA, USA) according to the user’s manual recommendations.
Typing
Separation of PCR products was carried out by capillary electrophoresis on an ABI PRISM1 3130xl Genetic Analyzer (Applied Biosystems). Genotyping was performed by comparison with sequenced allelic ladders provided with the kit, using the GeneMapper ID 3.2 software.
Quality control
Proficiency testing of the GEP-ISFG Working Group and PNCQ (‘‘Programa Nacional de Controle de Qualidade em Biologia Molecular’’, sponsored by the SBAC - Sociedade Brasileira de Análises Clínicas).
Analysis of data
Statistical analysis for allele frequencies, Power of Discrimination, Matching Probability, Polymorphism Information Content and Power of Exclusion were calculated using PowerStats v.13 software [3]. Deviation from Hardy-Weinberg equilibrium, Observed and Expected Heterozygosity was calculated using Arlequin v3.11 software [4].
Results
The allele frequencies and statistical parameters obtained for the 15 STR loci in Rio Grande do Norte are shown in Table 1.
Table 1: Allele frequencies and statistic parameters of forensic interest for 15 STR loci in the population of Rio Grande do Norte, Brazil.
ALELO D21S11 D13S317 D3S1338 VWA D7S820 D16S539 D18S51 CSF D5S818 TH01 TPOX FGA D8S1179 D2S1338 D19S433
6 0,223 0,023 7 0,011 0,012 0,034 0,231 0,001 8 0,098 0,140 0,031 0,022 0,016 0,146 0,455 0,006 9 0,086 0,118 0,174 0,019 0,036 0,175 0,117 0,006 0,001 9,3 0,217 10 0,054 0,298 0,081 0,007 0,234 0,080 0,008 0,055 0,069 0,005 11 0,288 0,001 0,001 0,230 0,273 0,012 0,312 0,320 0,293 0,063 0,022 11,2 0,001 12 0,301 0,004 0,177 0,273 0,125 0,325 0,326 0,056 0,143 0,106 12,2 0,013 13 0,127 0,005 0,005 0,023 0,148 0,120 0,062 0,174 0,271 0,260 13,2 0,001 0,043 14 0,044 0,095 0,079 0,003 0,020 0,156 0,010 0,011 0,260 0,001 0,286 14,2 0,001 0,033 15 0,002 0,304 0,118 0,001 0,167 0,003 0,002 0,143 0,002 0,133 15,2 0,048 16 0,251 0,272 0,133 0,035 0,036 0,032 16,2 0,013 17 0,203 0,283 0,124 0,001 0,003 0,214 0,001 17,2 0,001 18 0,128 0,155 0,069 0,009 0,095 0,001 18,2 0,002 0,001 19 0,009 0,067 0,048 0,065 0,122 20 0,018 0,019 0,112 0,136 21 0,002 0,009 0,160 0,050 22 0,005 0,141 0,072 22,2 0,003 23 0,001 0,146 0,112 23,2 0,002 24 0,159 0,072 24,2 0,001 0,000 25 0,001 0,122 0,065 26 0,001 0,050 0,021 27 0,023 0,001 0,013 0,002 28 0,153 0,005 29 0,217 0,002 30 0,261 0,001 30,2 0,025 31 0,052 31,2 0,104 0,002 32 0,014 32,2 0,102 0,001 33 0,001 33,2 0,026 34,2 0,003 0,001 35 0,003 36 0,009 43,2 0,001 45,2 0,001 n 2200 2200 2200 2200 2200 2200 2200 2200 2200 2200 2200 2200 2200 2200 2200 Ho 0.820 0,775 0,764 0,782 0,763 0,800 0,877 0,726 0,720 0,768 0,672 0,865 0,77 0,878 0,813 He 0.839 0.788 0,778 0,798 0,793 0,793 0,877 0,735 0,752 0,797 0,686 0,874 0,808 0,881 0,814 P <0,001 0.839 0,316 0,379 0,100 0,552 0,496 0,786 0,128 0,569 0,695 0,341 0,259 0,017 0,031 PD 0,954 0,927 0,917 0,933 0,928 0,924 0,971 0,884 0,901 0,929 0,856 0,970 0,939 0,974 0,940 PE 0,636 0,554 0,533 0,566 0,532 0,599 0,749 0,494 0,460 0,552 0,386 0,763 0,545 0,751 0,623 PIC 0.820 0,762 0,743 0,769 0,762 0,761 0,869 0,691 0,713 0,766 0,639 0,860 0,782 0,869 0,792 PI 2,780 2,227 2,115 2,292 2,107 2,500 4,074 1,930 1,786 2,216 1,522 3,691 2,174 4,104 2,670 BP 0,046 0,073 0,083 0,067 0,072 0,076 0,029 0,116 0,099 0,071 0,144 0,030 0,061 0,026 0,060 n, number of alleles; Ho, heterozygosity observed; He, heterozygosity expected; P, exact test probability for Hardy– Weinberg equilibrium; PIC, polymorphism information content; DP, discrimination power; MP, matching probability; TPI, typical paternity index; PE, power of exclusion.
Other markes
All the loci analysed reached the Hardy-Weinberg equilibrium in the population studied (P > 0.05), except D21S11 locus (P = 0.00007), D2S1338 (P = 0.017) and D19S433 (P = 0,031). When the Bonferroni correction [5] was employed using the number of loci analysed (P > 0.0033), the differences observed were not statistically significant, except for the D21S11 locus.
In the State of Rio Grande do Norte population, the observed heterozygosity (HO) ranges from 0.672 (TPOX) to 0.878 (D2S1338). The Power of Discrimination (PD) varies between 0.856 (TPOX) and 0.974 (D2S1338) and the probability of exclusion (PE) varies between 0.386 (TPOX) and 0.751 (D2S1338). The most polymorphic genetic markers is D18S51 and D2S1338 (both with PIC value of 0.8644) and the least polymorphic is TPOX (0.639).
The frequency distribution pattern of the 15 STR loci analyzed was compare to the ones obtained for other Brazilian populations [6-13], and we could find some rare alleles, as alleles 11,2 and 18 in D19S433 and allele 34,2 in FGA.
The comparison of the allelic frequency of this 15 loci studied in the population form Rio Grande do Norte State with others Brazilian populations [6-3] show that our population is genetically more proximal of the population of the State of Pernambuco [7] and genetically more distant of the population from the State of Amazonas [12] The forensic parameters regarding the 15 STR loci are useful in terms of variability, paternity and forensic purposes for the Rio Grande do Norte population.
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